PROJECT SUMMARY Tuberculosis (TB) is one of the leading infections in HIV/AIDS patients in the US. Globally TB continues to be a major public health threat with ~9 million new cases and 1.8 million deaths annually. Increasing number of cases reporting infection with multi- (MDR) and extensively-drug resistant (XDR) strains of M. tuberculosis (Mtb) has reduced our capability to respond effectively against this infectious disease. A major reason for emergence of drug resistance is thought to be poor compliance to treatment regimen as the existing therapy requires a combination of drugs to be taken daily for 6 months or more. While >99% of Mtb bacilli, the organism that causes TB, are killed within 2 weeks of therapy, it takes the remainder of the therapy to effectively kill the surviving population. These bacilli, broadly defined as [unreadable]Persisters[unreadable], are able to transiently tolerate drugs. Emerging evidence suggest that remodeling of the peptidoglycan layer with non-classical (3-->3) peptide linkages may be a mechanism by which Mtb survives in the hostile environment containing host generated anti-microbial products or in the presence of drugs used to treat TB. The peptidoglycan layer is an essential structure of Mtb and is maintained by 3-->4 and 3-->3 transpeptide linkages. The beta-lactams, the most widely used antibacterial drugs, target the 4-->3 linkages. However, Mtb is able to tolerate beta-lactams due to the presence of alternate 3-->3 linkages ( a mechanism different from that used by beta-lactamases). Recently we discovered a novel L,D-transpeptidase in Mtb that generates these 3-->3 linkages. We have shown that this gene, ldtMt2, previously annotated as a hypothetical encodes a L,D-transpeptidase that generates 3-->3 cross-linkages. We have also shown that drugs amoxicillin/clavulanate is active against Mtb lacking ldtMt2, illustrating that both 3-->3 and 4-->3 transpeptide linkages need to be destroyed to effectively kill M. tuberculosis. We propose to build on this finding to further characterize the enzyme, its pathway and exploit it to understand the mechanism of persistence by which Mtb survives in the host. The composition and structure of the peptidoglycan layer will be studied in the wild-type and mutant strains that lack L,D-transpeptidases at different stages of infection to determine physiological changes attributable to this novel mechanism for adaptation of the bacilli. We have determined the molecular structure of Ldt{Mt2} by solving its crystal structure. We will further study biochemical and biophysical properties of this enzyme and assess small molecule inhibitors to determine ways to inhibit it. This would be essential for development of drugs targeting L,D-transpeptidation to treat TB with a novel mechanism. Therefore, this proposal underscores the mission and priorities of NIH-NIAID.